To explore the effect of N-acetyltransferase（NAT） on the pathogenicity of Streptococcus agalacae， the nat gene deletion strain Δnat was constructed by homologous recombination in the background of a wild strain HN016. The growth curve， morphology， cell adhesion， invasion， antiphagocytosis， whole blood survival and pathogenicity of wild strain and Δnat strain were compared. The results showed that the hemolytic capacity of Δnat decreased， and the loss of nat decreased the adhesion and invasion of S. agalactiae to tilapia brain cells and increased the bacterial susceptibility to blood killing and phagocytosis. The result of challenge experiment of tilapia showed that the lethality of Δnat （2×10⁸ CFU/fish， the mortality rate was 36%） was significantly lower than that of wild strain HN016 （2×10⁸ CFU/fish， the mortality rate was 85%）. And the bacterial loads of Δnat were much less than that of HN016 in blood， spleen and brain tissue at 12 h post infection. This study firstly reports that NAT plays an important role in S. agalactiae resistance to host blood and phagocyte clearance， and assists in the bacterial invasion and colonization of tilapia tissues.